RESUMEN
Treatments with poxvirus vectors can have long-lasting immunological impact in the host, and thus they have been extensively studied to treat diseases and for vaccine development. More importantly, the oncolytic properties of poxviruses have led to their development as cancer therapeutics. Two poxviruses, vaccinia virus (VACV) and myxoma virus (MYXV), have been extensively studied as virotherapeutics with promising results. Vaccinia virus vectors have advanced to the clinic and have been tested as oncolytic therapeutics for several cancer types with successes in phase I/II clinical trials. In addition to oncolytic applications, MYXV has been explored for additional applications including immunotherapeutics, purging of cancer progenitor cells, and treatments for graft-versus-host diseases. These novel therapeutic applications have encouraged its advancement into clinical trials. To meet the demands of different treatment needs, VACV and MYXV can be genetically engineered to express therapeutic transgenes. The engineering process used in poxvirus vectors can be very different from that of other DNA virus vectors (e.g., the herpesviruses). This chapter is intended to serve as a guide to those wishing to engineer poxvirus vectors for therapeutic transgene expression and to produce viral preparations for preclinical studies.
Asunto(s)
Ingeniería Genética/métodos , Vectores Genéticos/genética , Poxviridae/crecimiento & desarrollo , Cultivo de Virus/métodos , Animales , Línea Celular , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Terapia Genética , Humanos , Poxviridae/genética , Transgenes , Células VeroRESUMEN
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide. Existing therapies for advanced tumors have high failure rates and can have severe consequences in terms of pain, disfigurement, and poor speech and swallowing function. New treatment strategies are needed to improve outcomes for patients suffering with this disease and oncolytic viruses represent a promising approach. METHODS: We infected six well-characterized HNSCC cell lines (Cal27, Detroit562, FaDu, SCC4, SCC15, SCC25), with increasing doses of a panel of poxviruses (including myxoma, vaccinia, raccoonpox and tanapox viruses) modified to express green fluorescence protein to determine which virus was the most effective oncolytic agent in cell-based assays. RESULTS: While myxoma, raccoonpox and tanapox displayed differing efficacy in the panel of cell lines, vaccinia virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all cell lines. CONCLUSION: Oncolytic poxviruses, particularly vaccinia virus, were effective in killing HNSCC in vitro and hold promise as potential treatments for patients with HNSCC.
Asunto(s)
Virus Oncolíticos/crecimiento & desarrollo , Poxviridae/crecimiento & desarrollo , Terapia Biológica/métodos , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Supervivencia Celular , Neoplasias de Cabeza y Cuello/terapia , HumanosRESUMEN
The double-stranded DNA genome of vaccinia virus (VACV), the prototype poxvirus, contains approximately 200 open reading frames (ORFs) that are transcribed at early, intermediate, and late stages of infection. Previous high-throughput deep RNA sequencing allowed us to map 118 VACV early genes that are expressed before viral DNA replication and 93 postreplicative genes. However, the intermediate- and late-stage postreplicative genes could not be differentiated. Here, we synchronized infections with a reversible inhibitor of DNA replication and used a VACV mutant that conditionally transcribes late genes to sequence the two classes of mRNAs. In addition, each postreplicative ORF was individually expressed under conditions that distinguished intermediate and late classes. We identified 38 VACV genes that belong to the late class and 53 that belong to the intermediate class, with some of the latter continuing to be expressed late. These data allowed us to prepare a genome-wide early, intermediate, and late transcription map. Inspection of sequences upstream of these ORFs revealed distinctive characteristics of intermediate and late promoters and suggested that some promoters have intermediate and late elements. The intermediate genes encoded many DNA binding/packaging and core-associated proteins in addition to late transcription factors; the late genes encoded many morphogenesis and mature virion membrane proteins, including those involved in entry, in addition to early transcription factors. The top-ranked antigens for CD4(+) T cells and B cells were mainly intermediate rather than late gene products. The differentiation of intermediate and late genes may enhance understanding of poxvirus replication and lead to improvements in expression vectors and recombinant vaccines.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Poxviridae/crecimiento & desarrollo , Poxviridae/genética , ADN Complementario/química , ADN Complementario/genética , ADN Viral/química , ADN Viral/genética , Genes Virales , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Viral/biosíntesis , ARN Viral/genética , Análisis de Secuencia de ADN , Transcripción Genética , Virus VacciniaRESUMEN
A synthetic route to (1S,2S,3R,5S)-3-(6-amino-9H-purin-9-yl)-5-fluorocyclopentane-1,2-diol (that is, the 4'-fluoro derivative of 4'-deoxy-5'-noraristeromycin, 3) is described via a fluorinated cyclopentanol, which is in contrast to existing schemes where fluorination occurred once the purine ring was present. Compound 3 was assayed versus a number of viruses. A favorable response was observed towards measles (IC(50) of 1.2 microg/mL in the neutral red assay and 14 microg/mL by the visual assay) but this was accompanied by cytotoxicity in the CV-1 host cells (21-36 microg/mL). Among the viruses unaffected by 3 were human cytomegalovirus and the poxviruses (vaccinia and cowpox), which are three viruses that were inhibited by the 4',4'-difluoro analog of 3 (that is, 2).
Asunto(s)
Adenosina/análogos & derivados , Antivirales/química , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Poxviridae/efectos de los fármacos , Adenosina/síntesis química , Adenosina/química , Adenosina/farmacología , Antivirales/síntesis química , Línea Celular , Citomegalovirus/crecimiento & desarrollo , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/farmacología , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Poxviridae/crecimiento & desarrollo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Camel pox viruses isolated in Sudan and VD45 (African camel pox strain) and Vaccinia virus (Elstree strain) were used for inoculation of chorioallantoic membrane (CAM) of embryonated eggs (EE) and cell culture (CC). In EE Lesions were seen as pocks ranging in size from 1 to 1.5 mm in diameter, and they increase in size with serial passage and taking opaque- white and opaque- yellow colors. When propagated in Vero cells, these viruses gave clear CPE, characterized by rounding of cells, plaque formation, syncytia and detachment of cells from the glass.
Asunto(s)
Membrana Corioalantoides/virología , Huevos/virología , Poxviridae/crecimiento & desarrollo , Virus Vaccinia/crecimiento & desarrollo , Células Vero/virología , Animales , Camelus , Embrión de Pollo , Chlorocebus aethiops , Membranas/virologíaRESUMEN
The seeding and working banks of a 4647-cell culture have been created. The 4647-cell culture in these banks has a high proliferative activity, as well as the morphology, typical of this line, and the karyotype and the enzymogram, which are characteristic for the cells of an African talapoin (Cercopithecus aethiops). The culture is not contaminated with bacteria, fungi, Mycoplasma, and viruses, including oncoviruses. The deposited 4647 cells have high viral productive properties for the accumulation of the recombinant virus strain b7,5S2-S vaccine and keep the stability of all biological properties during a long-term cultivation. The continuous 4647 cell line was tested at the L. A. Tarasevich State Institute of SK. The seeding and working banks of 4647-cell culture at passages 108 and 128 are recommended as a substrate for cultivation of the strain b7,5S2-S vaccinia, used to prepare a bivaccine against smallpox and hepatitis B.
Asunto(s)
Línea Celular/fisiología , Vacunas contra Hepatitis B/normas , Microbiología Industrial/normas , Vacuna contra Viruela/normas , Animales , Línea Celular/microbiología , Chlorocebus aethiops , Hepacivirus/crecimiento & desarrollo , Hepatitis B/inmunología , Cariotipificación , Poxviridae/crecimiento & desarrollo , Estándares de Referencia , Viruela/inmunología , Vacunas Sintéticas , Cultivo de Virus/normasRESUMEN
Enveloped animal viruses such as human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, human papillomavirus, Marburg, and influenza are major public health concerns around the world. The prohibitive cost of antiretroviral (ARV) drugs for most HIV-infected patients in sub-Saharan Africa and the serious side effects in those who have access to ARV drugs make a compelling case for the study of complementary and alternative therapies. Such therapies should have scientifically proved antiviral activity and minimal toxic effects. A plant extract, Secomet-V, with an anecdotal indication in humans for promise as an anti-HIV treatment, was investigated. Using a previously described attenuated vaccinia virus vGK5, we established the antiviral activity of Secomet-V. Chemical analysis showed that it has an acidic pH, nontoxic traces of iron (<10 ppm), and almost undetectable levels of arsenic (<1.0 ppm). The color varies from colorless to pale yellow to dark brown. The active agent is heat stable at least up to sterilizing temperature of 121 degrees C. The crude plant extract is a mixture of several small molecules separable by high-pressure liquid chromatography. The HIV viral loads were significantly reduced over several months in a few patients monitored after treatment with Secomet-V. Secomet-V was also found to have antiviral activity against the SARS virus but not against the West Nile virus. Secomet-V, therefore, is a broad-spectrum antiviral, which possibly works by neutralizing viral infectivity, resulting in the prevention of viral attachment.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , VIH/efectos de los fármacos , Extractos Vegetales/toxicidad , Poxviridae/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Trifolium , Proteínas Virales/toxicidad , Línea Celular , Humanos , Fitoterapia , Poxviridae/crecimiento & desarrolloRESUMEN
Poxviruses are cell-associated viruses that can be grown in adherent- or suspension-cell cultures or chorioallantoic membranes of embryonated hen's eggs. The main principle of isolating the virus is to mechanically lyse infected cells. The virus can then be semipurified by centrifugation through a sucrose cushion. Further purification can be achieved with a sucrose gradient. This chapter describes methods for the preparation of large-scale growth and purification of the virus.
Asunto(s)
Poxviridae/fisiología , Replicación Viral , Animales , Línea Celular , Embrión de Pollo , Poxviridae/crecimiento & desarrollo , Poxviridae/aislamiento & purificación , Cultivo de Virus/métodosRESUMEN
The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.
Asunto(s)
Antivirales/síntesis química , Herpesviridae/efectos de los fármacos , Nucleósidos de Pirimidina/síntesis química , Timina/síntesis química , Antivirales/química , Antivirales/farmacología , División Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Herpesviridae/crecimiento & desarrollo , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 2/crecimiento & desarrollo , Herpesvirus Humano 3/efectos de los fármacos , Herpesvirus Humano 3/crecimiento & desarrollo , Humanos , Cinética , Conformación Molecular , Poxviridae/efectos de los fármacos , Poxviridae/crecimiento & desarrollo , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Relación Estructura-Actividad , Timidina Quinasa/química , Timina/análogos & derivados , Timina/química , Timina/farmacología , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/crecimiento & desarrollo , Ensayo de Placa ViralRESUMEN
The study of infections of vertebrate animals by poxviruses has remained a dynamic area of research for the last century. The host range of poxviruses vary from extremely narrow to exceedingly broad, and they have been shown to enter their host by either the respiratory route or through the skin. The severity of infection varies dramatically from one species to another, causing anywhere from a local, self-limiting infection, to a devastating systemic disease, such as smallpox. Although the immune response to poxvirus infections are very similar to that seen in other viral infections, the poxviruses, unlike most other viruses (with the exception of Herpes viruses), are able to defend themselves. They have been shown to carry a repertoire of proteins involved in immune evasion and immune modulation. Poxviruses encode proteins involved in blocking many of the strategies employed by the host to combat viral infections; they encode for proteins that block activity of many chemokines, cytokines, serine proteases, and even complement. Traditionally, different animal models have been used to study the pathogenesis of poxvirus infections, and the characterization of virulence genes using mutant poxviruses. Additionally, new animal models are being developed to study the possible therapeutic uses many of these poxvirus immune modulating proteins might have. This review discusses the host immune response against poxvirus infections in various animals, the viral counter response to the host, and the animal models used to study poxvirus infection and immune modulating proteins.
Asunto(s)
Infecciones por Poxviridae/veterinaria , Poxviridae/patogenicidad , Animales , Anticuerpos Antivirales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Embrión de Pollo , Proteínas del Sistema Complemento/fisiología , Citocinas/inmunología , Modelos Animales de Enfermedad , Humanos , Inflamación/inmunología , Interferones/fisiología , Células Asesinas Naturales/inmunología , Ratones , Modelos Inmunológicos , Poxviridae/clasificación , Poxviridae/crecimiento & desarrollo , Infecciones por Poxviridae/complicaciones , Infecciones por Poxviridae/inmunología , Conejos , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/inmunología , Virus de la Viruela/crecimiento & desarrollo , Virus de la Viruela/inmunología , Virus/clasificación , Virus/inmunologíaRESUMEN
Vaccinia virus is being investigated as a replicating vector for tumor-directed gene therapy. However, the majority of cancer patients have preformed immunologic reactivity against vaccinia virus, as a result of smallpox vaccination, which may limit its use as a vector. The Yaba-like disease (YLD) virus was investigated here as an alternative, replicating poxvirus for cancer gene therapy. We have demonstrated that the YLD virus does not cross-react with vaccinia virus antibodies, and it replicates efficiently in human tumor cells. YLD virus can be expanded and purified to high titer in CV-1 cells under conditions utilized for vaccinia virus. The YLD virus RNA polymerase was able to express genes regulated by a synthetic promoter designed for use in orthopoxviruses. We sequenced the YLD virus TK gene and created a shuttle plasmid, which allowed the recombination of the green fluorescent protein (GFP) gene into the YLD virus. In a murine model of ovarian cancer, up to 38% of cells in the tumor expressed the GFP transgene 12 days after intraperitoneal virus delivery. YLD virus has favorable characteristics as a vector for cancer gene therapy, and this potential should be explored further.
Asunto(s)
Terapia Genética , Vectores Genéticos , Neoplasias Experimentales/terapia , Poxviridae/genética , Animales , Reacciones Cruzadas , Femenino , Ratones , Ratones Desnudos , Poxviridae/crecimiento & desarrollo , Poxviridae/inmunología , Regiones Promotoras Genéticas , Virus Vaccinia/genética , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/inmunología , Replicación ViralRESUMEN
A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Poxviridae/aislamiento & purificación , Animales , Técnicas de Cultivo de Célula , ADN Viral/análisis , Masculino , Poxviridae/genética , Poxviridae/crecimiento & desarrollo , Infecciones por Poxviridae/veterinaria , Sensibilidad y Especificidad , Ovinos , Enfermedades de las Ovejas/virología , Testículo/citologíaRESUMEN
Poxvirus was isolated from cutaneous nodules on two young Houbara bustards (Chlamydotis undulata) bred in captivity in Saudi Arabia. Birds were emaciated and presented nodules on tibiotarso-tarsometatarsal joints, toes, and the carpal joint. Diagnosis was confirmed by histopathology, virus isolation on inoculated chorioallantoic membranes of embryonated chicken's eggs, and electron microscopy. Progressive leg lesions were extensive and interfered with walking, significantly debilitating the birds. Successful excisions of these lesions were performed.
Asunto(s)
Enfermedades de las Aves , Viruela Aviar/patología , Enfermedades de la Piel/veterinaria , Animales , Aves , Embrión de Pollo , Articulaciones/patología , Microscopía Electrónica , Poxviridae/crecimiento & desarrollo , Poxviridae/aislamiento & purificación , Poxviridae/ultraestructura , Arabia Saudita , Piel/patología , Enfermedades de la Piel/patologíaRESUMEN
Dual infection with fowl pox (FP) and infectious laryngotracheitis (ILT) was diagnosed as the cause of acute mortality in a flock of three age groups of Hy-Line leghorn layers. The affected chickens had not been previously vaccinated against either FP or ILT. The diagnosis was confirmed by virus isolation, histopathology, and the use of specific pox and ILT genomic DNA probes in a dot-blot hybridization assay. FP and ILT vaccinations were recommended to control mortality. The use of FP- and ILT-specific DNA dot-blot hybridization may be used as a routine diagnostic tool to differentiate between the two diseases, especially in atypical cases of either infection or to confirm the existence of the two diseases as a mixed infection in a flock of chickens.
Asunto(s)
ADN Viral/análisis , Viruela Aviar/complicaciones , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1 , Enfermedades de las Aves de Corral , Alantoides/virología , Animales , Embrión de Pollo , Pollos , Corion/virología , Sondas de ADN , Viruela Aviar/diagnóstico , Infecciones por Herpesviridae/complicaciones , Infecciones por Herpesviridae/diagnóstico , Herpesvirus Gallináceo 1/genética , Herpesvirus Gallináceo 1/aislamiento & purificación , Hibridación de Ácido Nucleico , Poxviridae/genética , Poxviridae/crecimiento & desarrollo , Poxviridae/aislamiento & purificación , Mapeo RestrictivoRESUMEN
Polypeptide Ag35, a major early component of the vaccinia surface, is integrated into the formative viral lipoprotein tegument. To ascertain whether positioning of Ag35 is due to its general affinity for newly assembled viral membranes we created a recombinant A12 vector to express the vaccinia protein. The baculovirus system was chosen because intranuclear virions of this agent are likewise enclosed inside newly formed envelopes. Comparable infections of two insect cell lines established that more abundant synthesis occurred in High Five (H5) than in SF9 cells. We, therefore, used H5 cells for most experiments reported here. Combined analyses by PAGE, Western blotting, and immunocytology, using light and electron microscopy, revealed a dissemination of Ag35 throughout the cell. Higher concentrations were evident at the cell surface, nuclear perimeter, and within intranuclear virogenic stroma. The association with the virogenic stroma was of specific interest with respect to vaccinia development because it showed a similarity in the targeting of Ag35 toward intranuclear DNA-protein foci of baculovirus which are analogous to the vaccinia-specified cytoplasmic "factories." A further remarkable analogy concerns association of Ag35 with intranuclear baculovirus envelopes, revealing a propensity of Ag35 for nascent viral lipoprotein membranes.
Asunto(s)
Antígenos de Superficie/metabolismo , Antígenos Virales/metabolismo , Virus de Insectos/metabolismo , Poxviridae/metabolismo , Proteínas Virales/metabolismo , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/aislamiento & purificación , Antígenos Virales/genética , Secuencia de Bases , Transporte Biológico , Núcleo Celular/microbiología , Células Cultivadas , Citoplasma/microbiología , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Virus de Insectos/crecimiento & desarrollo , Virus de Insectos/ultraestructura , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Mariposas Nocturnas/ultraestructura , Nucleopoliedrovirus/genética , Poxviridae/crecimiento & desarrollo , Poxviridae/ultraestructura , Biosíntesis de Proteínas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Virión/crecimiento & desarrollo , Virión/ultraestructuraRESUMEN
Six isolates of erythromelalgia-related poxvirus (ERPV) were characterized with respect to host range, c.p.e. and inclusions, pock formation on chorioallantoic membrane (CAM), morphogenesis, serological reactivity, pathogenesis in animals and DNA restriction fragment profile. The results suggest that ERPV is either a new member of the Orthopoxvirus genus or a subspecies of ectromelia virus. Evidence is provided that (i) ERPV has a wide host range in vitro in which characteristic viral c.p.e. and inclusion bodies are induced; (ii) ERPV, unlike ectromelia virus, causes the formation of tiny greyish-white pocks on CAM both at 34 degrees C and 39 degrees C; (iii) eosinophilic A-type inclusions of ERPV do not contain viral particles; (iv) ERPV isolates are neutralized by both rabbit anti-vaccinia virus and mouse anti-ectromelia virus sera, but not vice versa; (v) young rabbits are not susceptible to ERPV by skin and/or corneal scratch infection even though ERPV is lethal for mice by intraperitoneal inoculation; (vi) the HindIII and SalI fragment profiles of ERPV P-4 DNA are similar to, but obviously different from, those of Chinese ectromelia virus. These biological and pathogenic characteristics of ERPV are distinguishable from those of other members of the genus Orthopoxvirus currently described in the literature.
Asunto(s)
Eritromelalgia/microbiología , Infecciones por Poxviridae/microbiología , Poxviridae/patogenicidad , Animales , Línea Celular , Reacciones Cruzadas , Efecto Citopatogénico Viral , ADN Viral/metabolismo , Humanos , Ratones , Morfogénesis , Pruebas de Neutralización , Poxviridae/genética , Poxviridae/crecimiento & desarrollo , Poxviridae/ultraestructura , Conejos , Ensayo de Placa ViralRESUMEN
A Saudi isolate of camel orthopoxvirus was serially propagated on monolayers of camel kidney cell cultures. The attenuation of the 78th passage was tested in two susceptible camels. Two other susceptible camels were inoculated with vaccinia virus four times propagated in camel kidney cell cultures. The four inoculated camels showed no postinoculation clinical symptoms and formed neutralizing antibodies against both the camel orthopox and vaccinia viruses. No postchallenge clinical symptoms were observed in these four camels, while two non-inoculated contact control camels showed typical symptoms of generalized camelpox. These results indicated the safety and potency of the 78th passage of the Saudi isolate of camel orthopoxvirus (designated Jouf-78) to be used for production of live attenuated cell culture camelpox vaccine. The field testing of the vaccine was carried out on two farms using at least 10(3) TCID50 as a recommended field dose. None of the inoculated camels showed any postvaccination reaction and the serological tests showed seroconversion of many vaccinated field camels. The relationship between camel orthopoxvirus and vaccinia virus as well as the advantages of the live attenuated camelpox vaccine are discussed.
Asunto(s)
Camelus , Poxviridae/inmunología , Vacunas Virales/inmunología , Animales , Camelus/microbiología , Células Cultivadas , Riñón/microbiología , Poxviridae/crecimiento & desarrollo , Vacuna contra Viruela/inmunologíaRESUMEN
Swinepox virus (SPV), the prototype member of the Suipoxvirus genus, is uncharacterized at the molecular level. We have analyzed the DNA of SPV and demonstrate that the genome is 175 kb in size and like the more commonly studied Orthopoxvirus, Avipoxvirus, and Leporipoxvirus genera, is terminally cross-linked and contains inverted terminal repetitions (ITRs). In addition, the ITRs are unstable, probably due to the presence of a variable number of direct repeats of approximately 70 bp in length. Restriction enzyme cleavage maps for the enzymes HindIII, AvaI, HaeII, KpnI, BglI, SalI, and XhoI are also presented.
Asunto(s)
ADN Viral/genética , Poxviridae/genética , Animales , Southern Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Genes Virales/genética , Peso Molecular , Hibridación de Ácido Nucleico , Poxviridae/crecimiento & desarrollo , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo RestrictivoRESUMEN
Three Vero cell culture-adapted contagious ecthyma virus (CEV) isolates were compared by plaque morphology, ability to induce vesicles in skin and in vivo growth curve characteristics by sampling sequentially experimental skin lesions produced in four sheep and one goat. Two of the isolates (CEV-29A and CEV-378) were from outbreaks of ecthyma in sheep and one (CEV-102) from a human case of orf. When replicating in Vero cells, the viruses exhibited similar growth parameters, but were distinguishable from each other on the basis of plaque morphology. In vivo latent periods for these isolates were 48 h (CEV-29A), 96 h (CEV-102), and 120 h (CEV-378). When isolates CEV-102 and CEV-29A were passaged into another sheep, they produced similar patterns of growth. Isolate CEV-102 produced the highest infectivity titer [1.4 X 10(9) plaque forming units (PFU) g-1], followed by CEV-29A (6.8 X 10(7) PFU g-1) and CEV-378 (2.5 X 10(7) PFU g-1). In addition, these viruses varied in their ability to induce vesicle formation. Virus was no longer detectable at the inoculation sites at 288 h post-infection (PI). We conclude that plaque morphology, ability to induce vesicle formation in the skin and growth curves in the skin can be considered as important criteria to differentiate CEV isolates. A comparison of the growth curves of CEV-378 in the skin of sheep and goats suggested differences in virus-host interaction between the two animal species. Since intravenous injection of 1 X 10(9) PFU of CEV failed to produce lesions in the sham-scarified skin of sheep, virus spread via the hematogenous route from one site to another appears unlikely. No virus-neutralizing antibody or interferons were found in serum samples or in skin homogenates collected between 0 and 24 days PI. Virus-neutralizing antibody was present in the circulation as late as 24 days PI. Lymphocytes collected from CEV-exposed sheep as early as 12 days PI responded specifically to stimulation with CEV antigen. As this was about the time when infectious virus disappeared from the sites, we assume that cell-associated immune mechanisms may play a larger role in virus clearance from skin lesions than virus-neutralizing antibody.